Aqueous Synthesis of PEGylated Quantum Dots with Increased

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Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity Mehriban Ulusoy,† Rebecca Jonczyk,† Johanna-Gabriela Walter,† Sergej Springer,‡ Antonina Lavrentieva,† Frank Stahl,† Mark Green,§ and Thomas Scheper*,† †

Institute of Technical Chemistry, and ‡Institute of Inorganic Chemistry, Gottfried Wilhelm Leibniz University of Hanover, , 30167 Hanover, Germany § Department of Physics, King’s College London, The Strand, WC2R 2LS London, U.K. S Supporting Information *

ABSTRACT: Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG− Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

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INTRODUCTION Colloidal nanoparticles (NPs) have become an important class of nanomaterials with potential for applications ranging from medicine to optoelectronic devices.1 The efforts of physical and material scientists to engineer NPs with unique material compositions and surface chemistries have led to tremendous progress in biological sciences to accomplish effective sensing, targeted delivery, imaging, and disease treatment.2 Despite all the work to exploit their unique characteristics in biomedical sciences, still little is known with respect to their interactions with complex biological environments.3 One of the dominating factors for NP interactions in their environments is the nature of their organic ligand coating (e.g., structure, ionic nature, packing density, hydrophobicity) that influences NP binding and uptake characteristics to a great extent.4 Even slight changes in surface functionalities might lead to greatly varying cellular internalization,5 indicating that the effect of NP surface physiochemical properties on their cellular interactions is far more complicated than currently acknowledged.6,7 When NPs are exposed to biological environments, they inevitably interact with serum proteins, which are adsorbed onto the NP surface, forming so-called “protein corona” structures.8 Adsorption of proteins to NP surfaces strongly affects the rate of NP adhesion © XXXX American Chemical Society

to cell membranes, which is one of the key determinants for NP uptake efficiency. For instance, lower adhesion due to the presence of biomolecular corona may cause a decrease in nanoparticle internalization or might induce specific recognition of proteins adsorbed to the NP by cell membrane receptors and therefore mediate NP uptake via receptormediated endocytic pathways.6,9 For in vivo applications, nonspecific interactions are not desired because they can hinder projected targeting and delivery; furthermore their accumulation can cause adverse effects by impairing membrane integrity.10 For that reason, engineering of NP surfaces to optimize physiochemical characteristics in order to reduce nonspecific interactions is a prerequisite to the application of NPs. Among various approaches, surface modification of NPs with polyethylene glycol (PEG) ligands has proved to be one of the most successful strategies.11 PEG ligands are nonbranched Special Issue: Molecular Imaging Probe Chemistry Received: September 8, 2015 Revised: November 4, 2015

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DOI: 10.1021/acs.bioconjchem.5b00491 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. (a) Schematic illustration of PEGylated CdTe/CdS/ZnS Qdots synthesis in aqueous medium; (b) red-shift in emission profiles of Qdots during ZnS shell growth in the presence of mPEGthiols; (c) full-width at half-maximum (fwhm) values for resulting Qdots as an indicator for size variations. 3-Mercaptopropionic acid (MPA), which was used as sulfur source and at the same time as surface passivating ligand, coordinated at outer surface along with thiol-terminated mPEG molecules during ZnS shell growth.

limiting factor for their grafting density. However, the in situ approach can enable a tight surface coverage, thereby providing greater efficiency in preventing protein adsorption.27 Various in situ approaches for the synthesis of colloidal NPs in the presence of PEG molecules have been accomplished in previous studies. For instance, Mukhopadhyay et al. reported the synthesis of PEG-coated magnetite (Fe3O4) NPs to investigate their effect on cytochrome c reduction.28 Seol et al. developed a one-step, microwave-assisted route for the synthesis of PEG-coated gold nanoparticles (GNPs).29 Shameli et al. facilitated PEG-mediated growth of silver nanoparticles (Ag-NPs) while applying an environmentally friendly approach.30 Huang et al. managed a controllable large-scale synthesis of ultrasmall PEG−ZnO NPs in ethanol.31 Moreover, Shen et al. presented one-pot hydrothermal synthesis of PEGpassivated graphene quantum dots and also studied their photoelectric conversion under near-infrared light.32 Recently, Yinan et al. developed water-soluble Mn2+-doped ZnS nanocrystals with PEG as a surface modifier without a ligand exchange protocol to render the NPs water-soluble.33 Rao et al. introduced simultaneous synthesis and surface functionalization of BaLuF5:Gd/Yb/Er upconversion NPs (UCNPs) with PEG ligands as an ideal probe for dual imaging.34 Despite all these successful synthesis attempts for NPs in the presence of PEG,

polymers that have high exclusion volumes due to their high conformational entropy, so as a result, they repel biomolecules.12 Moreover, they provide colloidal stability via steric repulsion and introduce higher flexibility to NP surfaces; they have high resistance to protein adsorption and therefore reduce nonspecific NP−cell membrane interactions.13−15 Their hydrophilic and nontoxic nature also mitigates NP-induced adverse effects by preventing chemical degradation of NPs and release of their metal ions to intracellular environments.16,17 They also hinder recognition of NPs by the immune system, thus reducing NP clearance by the reticuloendothelial system (RES) and increasing retention times for NPs in blood circulation.17,18 It has already become clear that PEGylation density and the PEG spatial configuration have a great impact on its physiological properties. The amount and configuration of grafted PEG play determinant roles in NP colloidal stability, protein adsorption, and cellular internalization.11,19,20 It has also been reported that densely packed, planar, PEGylated surfaces can suppress protein adsorption only in certain configurations.21 PEG molecules can be linked to NP surfaces either via incorporating PEG chains in situ11,22 or through covalent attachment to the surface.23−26 When PEG polymers are covalently attached to surfaces, their chain entropy becomes the B


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was increased to 1:10 in the reaction solution, ZnS shell deposition rate decreased drastically, with a subsequent decrease in quantum yields (QY, %) by 3 ± 1%. For the growth of the ZnS shell, sulfur atoms from the Cd-SR on the surface of CdTe/CdS Qdots released their organic group R = [−(CH2)2COOH] to bind with Zn2+ ions. Unreacted MPA molecules in the crude solution also bound with Zn2+ ions to form an outer shell as well as to coordinate on the shell surface.40 In the meantime, mPEGthiol molecules attached to the ZnS shell surface by means of zinc−thiol binding. In the presence of excess mPEGthiols, the binding of the sulfur atoms of mPEGthiols to the Qdot surface might have taken place at the early stage of ZnS growth. Consequently, they might have hindered formation of Zn−MPA complexes, thus slowing deposition of the ZnS shell. Fluorescence emission peak widths (full-width at half-maximum, fwhm) of the resulting Qdots are given in Figure 1c. COOH−Qdots, mPEG−Qdots (1:5), and mPEG−Qdots (1:10) showed very similar fwhm values at 72.9 ± 3.1, 74.2 ± 2.5, and 74.1 ± 1.7, respectively. The spectral width values indicated that there was no significant difference in Qdot size distributions for the samples. That is to say, the applied PEGylation strategy did not cause a broadening of size distribution; as a result, Qdots preserved their monodispersity. Characterization Studies. ζ-potential (ζ-Pot) analyses were conducted in order to measure the charge of the Qdots. COOH−Qdots with negatively charged carboxyl groups on their surface had a negative ζ-Pot value of −36.8 mV (Figure 2a). Meanwhile, mPEG-capped Qdots featured reduced ζ potential values due to the partial replacement of carboxyl groups with nonionic mPEG molecules. mPEG−Qdots (1:5) showed ζ-Pot value of −31.1 mV, whereas mPEG−Qdots (1:10) showed −25.9 mV; thus, as the amount of mPEG molecules on nanoparticle surface was increased, the corresponding ζ-Pot value was reduced further. Although we lack the knowledge of the precise number of mPEG molecules attached per Qdot and their orientation on the surface, the ζpotential analysis clearly indicates that mPEG−Qdot (1:10) conjugates possessed a higher degree of mPEG coating in comparison to mPEG−Qdots (1:5). Attenuated total reflection Fourier transform infrared (ATRFTIR) spectroscopy results for COOH−Qdots and mPEG− Qdots (1:10) are given in Figure 2b. In general, vibration frequencies exhibited slight shifts for mPEG−Qdots in comparison to COOH−Qdots. According to the spectra, symmetric and asymmetric −COO stretching bands for COOH−Qdots were observed at 1404 and 1560 cm−1, respectively,41 while for mPEG−Qdots they were shifted to 1402 and 1549 cm−1 with a concomitant decrease in their intensity. The O−H stretching bands (3412 cm−1) and C−OH stretching peak (1140 cm−1) for COOH−Qdots appeared at 3425 and 1103 cm−1, respectively, for mPEG−Qdots, and both experienced a decrease in their intensity. At the same time, the −OH bending peak for carboxylic acids (933 cm−1) was shifted to 947 cm−1 for mPEG−Qdots, with a significant decrease in its intensity. These decreases can be attributed to partial replacement of −COOH groups with mPEG molecules. Moreover, C−H stretching peak of alkyl group (2937 cm−1) for COOH−Qdot was moved to 2924 cm−1 for mPEG−Qdot with a significant increase in its intensity. This increase can be attributed to the presence of terminal methyl groups (−CH3) and −CH2 groups of mPEG backbone. Thermogravimetric (TG) analysis was conducted to estimate the amount of mPEG molecules on the surface of Qdots. As

the effects of PEG molecules on the generation of NPs and their physiochemical properties have not been thoroughly investigated. In this study, we present a practical and generic approach for the in situ introduction of thiol-terminated methoxy PEG (mPEG) groups (Mn = 800) to the surface of CdTe/CdS/ZnS Qdots during synthesis of ZnS outer shell, with simultaneous incorporation of surface carboxyl groups for further functionalization. Further, we investigated the effect of mPEG ligands on the physiochemical properties of Qdots in terms of shell growth rate, surface characteristics, and colloidal stability. In our previous report, we established aqueous synthesis of nearinfrared emitting CdTe/CdS/ZnS core (small) /shell (thick) / shell(small) Qdots with high quantum yields up to 64%. There, we indicated that ZnS shell deposition minimized Qdotinduced in vitro cytotoxicity and increased photostability.35 Although ZnS is less susceptible to oxidation than CdS, the relatively slow oxidation of ZnS with a concomitant loss of surface sulfur atoms can take place under ambient conditions.36 This process eventually results in the decomposition of the ZnS shell and brings delayed-release Qdot toxicity issues back into the discussion. Taking this into consideration, we tested the effect of mPEG surface ligands on the cellular toxicity of Qdots as a function of mPEG grafting density by using twodimensional (2D) monolayer cell cultures and three-dimensional (3D) tumor-like spheroid cultures. Later, we explored cellular binding characteristics of PEGylated and nonPEGylated Qdots with 3D spheroids in order to understand their interactions on physiologically relevant microscale tissue-like structures to predict in vivo behavior of Qdots in the sense of cellular binding and uptake. We believe that the result of this study will highlight fundamental parameters that need to be explored in detail to boost the development and safe application of functionalized colloidal NPs in nanobiotechnology.

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RESULTS AND DISCUSSION Synthesis of PEGylated CdTe/CdS/ZnS Qdots. Aqueous one-pot synthesis of 3-mercaptopropionic acid (MPA)functionalized CdTe/CdS/ZnS Qdots with small-core/thickshell/thin-shell structure was adopted from our previous study.35 First, CdTe/CdS small-core/thick-shell Qdots emitting at 650−660 nm were synthesized. The crude solution was directly used for deposition of a ZnS shell, and the remaining MPA molecules in the crude solution were utilized as the sulfur source for ZnS formation as well as a surface-coordinating agent. In addition to MPA, methoxy PEG thiol (mPEGthiol) molecules were introduced to the surface of the outer ZnS shell as a cocoordinating ligand. Thus, CdTe/CdS/ZnS Qdots with a mixture of carboxyl and mPEG surface groups were created. A schematic representation of ZnS shell overgrowth in the presence of MPA and mPEGthiols is depicted in Figure 1a. The amount of mPEGthiol in the reaction solution was adjusted to give a [Zn/mPEGthiol] molar ratio of 1:5 and 1:10. The resulting mPEG−Qdots are respectively referred to as mPEG− Qdot (1:5) and mPEG−Qdot (1:10) elsewhere in the text. During the ZnS shell synthesis, fluorescence emission profiles showed red-shift indicating the deposition of a ZnS shell.35,37−39 However, the amount of red-shift declined in the presence of mPEGthiol molecules (Figure 1b), indicating a slower growth of the outer ZnS shell. For mPEG−Qdot (1:5), ZnS shell formation proceeded slightly slowly without any effect on Qdot fluorescence efficiency. As the amount of mPEG C


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able weight loss at higher temperatures between 500 and 680 °C which was related to the degradation of mPEG molecules. In addition to that, the drop between 300 and 525 °C was found to be pronounced for mPEG−Qdots in comparison to COOH−Qdots, suggesting that decomposition of mPEG molecules had already started at low temperatures, which is in agreement with previous reports.44−46 Regarding the organic capping materials, COOH−Qdots exhibited a weight loss of 21.3% whereas mPEG−Qdots (1:5) and mPEG−Qdots (1:10) showed a 34.4% and 41.7% weight loss, respectively. The increase in the amount of weight loss for the PEGylated Qdots corresponds to an increase in the amount of mPEG molecules on the surface of the Qdots. The corresponding mPEG amounts are approximately 13.1% and 20.4% for mPEG−Qdots (1:5) and mPEG−Qdots (1:10), respectively, suggesting that mPEG−Qdots (1:10) acquire 27% excess mPEG molecules in comparison to mPEG−Qdots (1:5). As a conclusion, these results indicate successful attachment of mPEG groups to the Qdot surfaces during ZnS shell synthesis. Colloidal Stability. Ligand molecules on Qdot surfaces play an important role on colloidal stability of NPs by influencing their nanoenvironments.4 3-Mercaptopropionic acid (MPA), the main surface coordinating ligand for Qdots used in this study, plays a major role that determines the colloidal stability of Qdots. When Qdots are dispersed in pure water, carboxyl groups of MPA become negatively charged, since in pure water pH (7.0) ≫ pKCOOH (4.32).47 As a result, the NP surface becomes saturated with negatively charged (−COO−) groups and the NPs become colloidally stable due to the electrostatic repulsion between negative charges. In addition to electrostatic repulsion, NPs can also be stabilized via steric repulsion originating from macromolecular ligands, such as PEG.4 Herein, we studied the colloidal stability of carboxylated and PEGylated Qdots in order to examine the cooperative influence of neutral mPEG molecules on Qdot stability. As is known, aggregation of Qdots in aqueous solution results in a red-shift in emission profiles with a subsequent decrease in their fluorescence intensity.31,47,48 Considering this, changes in emission wavelength positions for respective Qdots upon their incubation in different aqueous media at different temperatures were established as an indicator of their colloidal stability. For that, diluted Qdots solutions (≤1 mg mL−1) were prepared in pure water, serum-free culture medium, and 10% serum-containing medium and incubated at 4 and 37 °C. The obtained results are depicted in Figure 3. Accordingly, both COOH−Qdots and mPEG−Qdots showed different levels of stability with regard to their mPEG content. As a general observation, COOH−Qdots showed a greater tendency for aggregation in all aqueous media tested in comparison to their PEGylated counterparts, and a high incubation temperature induced faster Qdot agglomeration. As a result, the red-shift in COOH−Qdots emission reached a plateau earlier. In pure water, protonation of the thiol ligands of MPA may lead to an ultimate loss of surface agents.49 The diluted Qdot concentration plays a major role in the irreversible loss of MPA due to the impaired dynamic equilibrium of detaching and rebinding of MPA.50 Once the surface capping agent is detached from the surface, electrostatic repulsion forces become insufficient to ensure the stabilization of Qdots; in addition to that, free inorganic surface energy rises and in turn triggers interparticle attractions by van der Waals forces. This mechanism might be one of the main causes of the loss of colloidal stability in thiol-

Figure 2. Characterization of COOH− and mPEG−Qdots: (a) ζpotential analysis; (b) ATR-FTIR spectra of (i) COOH−Qdot and (ii) mPEG−Qdot (1:10) films casted on a ZnSe ATR crystal in water; (c) TG analysis of COOH−Qdots and mPEG−Qdots. The heating rate was 5 K min−1 under an 80% argon and 20% O2 flow. The vertical line indicates the temperature at which most of the adsorbed water was considered to be evaporated (175 °C).

shown in Figure 2c, COOH− and mPEG−Qdots displayed different amounts of weight loss between 175 and 680 °C with respect to their surface ligand structure. The drop in all TG curves until 175 °C was attributed to the decomposition of adsorbed water. Between 175 and 525 °C all Qdot samples exhibited a similar mass-loss profile which was assigned to the gradual decomposition of MPA ligands from the surface of the Qdots.42 The decomposition temperature of MPA, which is higher than the boiling point of pure MPA (157 °C), demonstrates covalent bonding between MPA and the Qdot surface.43 Meanwhile, mPEG−Qdots displayed a distinguishD


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Figure 3. Structural stability of COOH−Qdots and mPEG−Qdots in water, DMEM cell culture medium with/without 10% fetal calf serum (FCS) at 4 and 37 °C. Graphics show red-shift in Qdots emission wavelengths versus incubation time in given aqueous media. The aggregation induced redshift in Qdot emission shows different profiles regarding the mPEG content.

temperature dependency, and the temperature effects were pronounced at the physiologically relevant temperature at 37 °C. At physiological temperatures, structural fluctuations of proteins and/or polymer shells around Qdots might induce conformational arrangements which in turn might affect the binding affinity of proteins to the Qdot surface.52 Therefore, the decreased agglomeration behavior for Qdots in serumcontaining medium at 37 °C could be accounted for by a higher degree of protein adsorption to Qdot surfaces than at 4 °C. Overall, the obtained results proved that higher amount of mPEG molecules on the surface of ZnS shell enhanced colloidal stability of Qdots via steric repulsion effects to a great extent. Effect of mPEG on Protein Adsorption. The influence of PEGylation on protein adsorption was further investigated by protein interaction analysis using bovine serum albumin (BSA) as a model protein prevalent in serum. We expect that mPEG− Qdots should possess a reduced degree of protein binding and thus a decreased protein corona formation. In order to prove this assumption, microscale thermophoresis (MST) analysis of Qdots and BSA was performed, and equilibrium dissociation constants determined for COOH− and mPEG−Qdots are given in Figure 4.53 Hill model was used to extract quantitative data about binding affinity of BSA to the surface of Qdots with regard to their mPEG content.50,51,54 By fitting experimental data with Hill equation, we obtained apparent equilibrium dissociation coefficient Kd values (concentration of protein at half saturation) of Kd = 0.306 ± 0.002 μM for COOH−Qdots, Kd = 0.406 ± 0.016 μM for mPEG−Qdots (1:5), and Kd = 0.771 ± 0.077 μM for mPEG−Qdots (1:10). Accordingly, binding affinities were found to decrease for Qdots in the following order: COOH−Qdots > mPEG−Qdots (1:5) >

capped Qdots. Incorporation of neutral mPEG polymers to the Qdot surface facilitated a significant improvement in overall colloidal stability by introducing a steric barrier to prevent interparticle interaction. When compared to other Qdots, mPEG−Qdots (1:10) possessing the highest surface mPEG amount had the highest colloidal stability. Increased grafting density might have provided a more stable brush layer through coordination of the thiol functional group with Zn2+ ions during ZnS shell growth. In serum-free culture medium, the presence of salt cations (e.g., Na+ and Ca2+) expedited aggregation of all Qdots, especially that of carboxyl-capped Qdots. The underlying reason is that the negative surface charges of carboxyl groups are screened by the absorption of counterions from culture medium. Due to the screening effect, electrostatic repulsive forces were reduced. As a result, interparticle accumulation became favorable.50 In serumcontaining culture medium, all Qdots emissions exhibited smaller red-shifts in comparison to serum-free medium conditions, implying relatively higher colloidal stability in serum-containing medium. This can be accounted for by the formation of protein corona structures in the presence of serum proteins.9,50 Adsorption of a protein layer on colloidal NP surfaces has proven to improve the stability of NPs against agglomeration.51 The degree of protein corona formation is closely related to the physiochemical properties of nanoparticles (e.g., size, charge, surface chemistry, colloidal stability).3 In the presence of serum proteins, formation of a protein corona should predominate Qdot agglomeration rather than interparticle attractions alone. In serum-containing medium at 4 °C, Qdots reached a plateau later than at 37 °C, suggesting that adsorption of proteins displayed a marked E


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amount caused a further decrease in the interactions of the Qdots with BSA proteins, confirming our expectation. The Hill coefficients were also determined to interpret the degree of cooperativity in protein binding to the surface of the Qdots. The Hill fit yielded a Hill coefficient of n = 2.0 ± 0.1 for COOH−Qdots, n = 3.1 ± 0.7 for mPEG−Qdots (1:5), and n = 2.6 ± 0.3 for mPEG−Qdots (1:10), indicating a cooperative binding behavior (n ≥ 1) for all Qdots. That is to say, the proteins bound on the Qdots surfaces influenced the adsorption of further proteins from the solution. The cooperative adsorption of proteins to NP surfaces has been reported before and was attributed to stabilizing interactions between adjacent protein molecules that may contribute to formation of protein corona.51,55 Evaluation of in Vitro Cytotoxicity. The rapid emergence of quantum dots as highly efficient biological imaging agents brought along safety concerns related to their metal-containing compositions.56 NP-induced toxicity is mostly ascribed to in situ NP degradation which is followed by release of metal ions (e.g., Cd2+, Ag+, Pb2+, In3+, As3−, Hg2+, Au+) to the intracellular environment. After their internalization, NPs are entrapped in endocytic vesicles and undergo a surface etching process under acidic conditions in lysosomes, which results in irreversible destruction of their photochemical properties.57−59 NP-induced toxicity exhibits a direct relation to their material composition, size, surface chemistry, and colloidal stability. In our previous

Figure 4. Determination of the dissociation constants of Qdots−BSA interactions by MST analysis. Kd values were calculated by fitting experimental data with Hill equation. Samples were excited at 470 nm with an excitation power of 10%, and thermophoresis analyses were conducted at room temperature with a 40% MST power. The error bars represent the standard deviation of at least two independent MST measurements.

mPEG−Qdots (1:10). These results indirectly prove a higher amount of grafted mPEG for mPEG−Qdots (1:10) than that of mPEG−Qdots (1:5) and indicate that an increase in the mPEG

Figure 5. (a) Dose-dependent in vitro cytotoxicity of COOH−Qdots and mPEG−Qdots on A549 cell lines; (b) cell viability data obtained from CTB assay for 2D cell cultures; (c) bright-field microscope images of A549 tumor-like spheroids exposed to COOH− and mPEG−Qdots (1:5) with a concentration range of 0.002−1000 μg mL−1 for 24 h. Data points are given as mean ± standard error of 3 ± 1 independent experiments of which each was conducted with four replicates. Inset data in (a) show half-maximal inhibitory concentrations (IC50) in μg mL−1. The asterisk (∗) indicates significant difference at 0.01 level. p < α = 0.01 (ANOVA one-way). Scale bars in (c) are 200 μm. F


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of ZnS shells may have an influence on overall Qdot-induced toxicity. Nevertheless, despite the restricting effect of mPEGthiol molecules on formation of a well-passivated ZnS shell, the improved colloidal stability of Qdots predominantly prevented aggravation of Qdot toxicity. On 3D spheroids, the general decrease in Qdot-induced toxicity in comparison to 2D cultures can be explained by the presence of a physical barrier arising from extracellular matrix (ECM), which is produced by the cells during the spheroidal growth.67 This barrier can hinder NP accumulations and limit their transport toward the spheroid inner region. Such physical limitations are not as pronounced in 2D monolayer cultures as they are in 3D models.68 Supporting our findings, conventional monolayer cell cultures were also reported to be more sensitive to NPs or drug molecules in comparison to various 3D cell culture systems in previous studies.63,64,69−71 Nonspecific Cellular Binding. Nonspecific binding features of COOH−Qdots and mPEG−Qdots with A549 cells were analyzed by using flow cytometry measurements. Fluorescence intensities were obtained from cells that had been incubated with Qdots (Figure 6). Consequently, COOH−

study, we addressed size and composition-dependent adverse effects of COOH−CdTe/CdS and COOH−CdTe/CdS/ZnS Qdots on monolayer cultures of A549 human lung adenocarcinoma cells. 35 The results demonstrated that deposition of an outer ZnS shell mitigated the toxic effects of Qdots by providing effective surface passivation. Herein, we aim to address the subsequent effect of mPEG molecules grafted to ZnS shell surfaces on Qdot-induced toxicity as a function of mPEG density. Additionally, 3D tumor-like spheroid cultures of A549 cells were utilized in order to assess physiologically relevant cytotoxicity effects of PEGylated Qdots. Spheroid cell cultures are a relatively simple technique of 3D cell cultures that takes advantage of the natural tendency of cells to aggregate.60 They provide a more accurate in vitro model, since they closely resemble in vivo tissue structures in terms of intercellular communication, extracellular matrix development, and complex diffusion/transport mechanisms in comparison to 2D monolayer cell cultures. For that reason, the utilization of 3D cell culture models holds great potential to produce more physiologically relevant data for NP-based studies.61−64 The cell viability results obtained from CTB assay are given in Figure 4. In 2D monolayer cultures, PEGylated Qdots exhibited significantly reduced toxicity in comparison to carboxylated Qdots (Figure 5a). Half-maximal inhibitory concentrations (IC50) were calculated to be 152.1 ± 21.4 μg mL−1 for COOH−Qdots and 282.9 ± 26.4 μg mL−1 for mPEG−Qdots (1:5), thus meaning ∼86% decrease in cellular toxicity upon incorporation of mPEG molecules to Qdot surface. On the one hand, mPEG−Qdots (1:10) possessing more mPEG molecules resulted an IC50 value of 286.5 ± 26.4 μg mL−1 which is very close to that of mPEG− Qdots (1:5). For 3D tumor-like spheroid cultures, all Qdot samples tested demonstrated reduced toxicity levels for the applied Qdot doses in comparison to 2D cultures (Figure 5b). PEGylation of Qdots mitigated dose-dependent adverse effects on 3D spheroids, agreeing with the cell viability results obtained from 2D cultures. Also, increased mPEG quantities on Qdot surface did not lead to any further improvement on Qdots toxicity (data not shown). According to the bright-field images of spheroids shown in Figure 5c, Qdots induced apparent effects to the morphology of tumor-like spheroids in a concentration-dependent manner. Low Qdot concentrations (99.8%), trisodium citrate dehydrate (HOC(COONa) (CH2COONa)2· 2H2O), polyethylene glycol methyl ether thiol (mPEGthiol, average Mn = 800), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), Calcein-AM (BioReagent, ≥96.0%), and bovine serum albumin (BSA, heat shock fraction, pH 7, ≥98%) were purchased from Sigma-Aldrich GmbH, Munich. CellTiterBlue cell viability assay kit was purchased from Promega Corp., USA. For cell culture experiments, Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich GmbH, Munich) supplemented with 10% (v/v) fetal calf serum (FCS, Biochrom GmbH, Germany) and 1% (v/v) penicillin/streptomycin (P/S, Biochrom GmbH, Germany) was used as cell culture medium. For 2D cell cultures, 96-well flat bottom standard plates (Sarstedt AG & Co., Germany) were used. For 3D spheroid I


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Bioconjugate Chemistry FTIR spectra were collected for Qdots films (2 mg mL−1) casted on ZnSe crystal surface. Thermogravimetric (TG) analyses for powder Qdot samples (∼5 mg) were performed using a STA 409 PC/PG apparatus (Netzsch, Germany) under 80% argon and 20% O2 flow (flow rate: 100 mL min−1) with a heating rate of 5K min−1. Stability Test. To test colloidal stability of Qdots, 1 mg mL−1 of Qdots solutions (250 μL) were prepared in pure water, DMEM culture medium, and DMEM culture medium supplemented with 10% FCS. Samples prepared in each medium were incubated at 4 and 37 °C to study the effect of temperature on colloidal stability. Emission wavelengths of each sample were recorded at every 24 h for 9 days. Microscale Thermophoresis (MST) Analysis. The equilibrium dissociation constants (Kd) of Qdot−BSA interactions were measured via MST analysis. Qdot concentration was kept constant at 100 μg mL−1, and the concentration of BSA was varied from 0.013 μg mL−1 to 412.5 μg mL−1. After the samples were incubated at room temperature for 5 min, they were loaded to standard treated glass capillaries (NanoTemper Technologies GmbH, Munich, Germany) for MST measurements. The experiments were performed with a Monolith NT.115 MO-G008 (NanoTemper Technologies GmbH, Munich, Germany) using 40% MST power, and the samples were excited at 470 nm with 10% excitation power. NT Analysis software (NanoTemper Technologies GmbH, Munich, Germany) was utilized for fitting the curves by using Hill model to determine the Kd values. Cytotoxicity Studies. For the in vitro cytotoxicity studies, human lung adenocarcinoma A549 cell line (ACC107) purchased from DSMZ (German Collection of Microorganism and Cell Cultures) was used with a passage number of less than 20. For assessment of cytotoxicity on 2D cultures, 8000 cells in 100 μL of culture medium (DMEM supplemented with 10% FCS and 1% P/S) were seeded to 96-well flat bottom standard plates and incubated for 2 days at 37 °C, 5% CO2. Qdot dilution series were prepared with cell culture medium containing supplements. An amount of 100 μL of culture medium was replaced with Qdot-containing medium, and cells were incubated for the next 24 h. A control group was treated with fresh culture medium without Qdots. For utilization of 3D spheroid cultures, 6000 cells in 100 μL of culture medium were seeded to 96-well round-bottom spheroid plates and incubated for 2 days for spheroid formation. Afterward, 50 μL of culture medium was replaced with 50 μL of Qdot solution freshly prepared with culture medium and incubated further for another 24 h. The metabolic activity of viable cells in terms of their reduction capacity of resazurin was measured via CTB assay kit (Promega Corp., USA). For 2D cell culture, culture medium was removed gently, and 100 μL of CTB reagent (diluted 1:6 with supplement-free DMEM medium) was added to each well and incubated for 1−2 h (37 °C, 5% CO2). For 3D spheroid cultures, 20 μL of CTB stock reagent was directly added to 100 μL of Qdot-containing culture medium 5 h after Qdot introduction (giving a final dilution of 1:6) and incubated for another 19 h at 37 °C, 5% CO2. The resulting fluorescence intensities originated from release of resorufin dye were recorded at 544Ex/590Em with fluorescence spectrometer (Fluoroskan Ascent, Thermo Fischer Scientific Inc. USA). Statistical Analysis. Concentration-dependent normalized cell viability data obtained from CTB assay were fitted from 0 to 100 by using nonlinear curve fitting/growth/sigmoidal/ dose−response fitting functions (OriginPro 8.6.0 b70, Origin-

Lab Corp., USA). Half-maximal inhibitory concentrations (IC50) were calculated from the fitted dose−response curves. The shown data are from at least two independent experiments, and all individual experiments were conducted with four replicates (n = 4). Levene’s test to assess the homogeneity of variance of replicates and then one-way analysis of variance (ANOVA) for the comparisons of mean values of independent groups were performed at the level of 0.01 (α = 0.01) (OriginPro 8.6.0 b70). A significant effect was reported at (∗) p < α (0.01). Flow Cytometry Studies. Nonspecific binding of Qdots was analyzed quantitatively by using a COULTER EPICS XLMCL flow cytometer. A549 cells were first harvested with Accutase solution (Sigma-Aldrich). Following centrifugation at 300g for 5 min, cell suspension was collected, and the number of viable cells was determined by trypan blue exclusion. Later, A549 cell suspensions (5 × 105) were incubated with 100 μg mL−1 COOH−Qdots and mPEG−Qdots prepared in culture medium (DMEM supplemented with 10% FCS and 1% P/S) for 30 min at 26 °C/300 rpm. Afterward cells were washed three times with PBS and resuspended in 500 μL of PBS buffer (supplemented with 2% FCS). Cells were filtered through cell strainer (BD Falcon, 70 μm nylon) directly into capillary tubes for flow cytometry measurements. An excitation laser at 488 nm was used, and corresponding fluorescence intensities were collected with the fluorescence-3 (FL3) sensor using band-pass filter at 675 ± 15 nm, and the data was analyzed by WinMDI 2.9 software. Cellular fragments and debris were excluded from the analysis by gating the fluorescence on the side scatter vs forward angle light scatter signal. Geometric-mean (g-mean) fluorescence intensity of the cellular autofluorescence was subtracted from the g-mean values of the positive cells. The binding effect was defined as the ratio between the normalized fluorescence intensity of cells incubated with COOH−Qdots and mPEG−Qdots. Microscopy Studies. Bright-field images of A549 tumorlike spheroids that were treated with Qdots at different concentrations were captured with Olympus Ix50 inverted light microscope equipped with an Olympus camera (SC30, Japan) by using cellSens Standard software (Olympus Co. Japan). For labeling experiments, A549 spheroids were treated with 100 μg mL−1 Qdots solutions for 1 and 24 h at 37 °C, 5% CO2. After incubation, spheroids were washed gently with PBS solution and placed on μ-slide glass bottom microscopy chamber slides (ibidi GmbH, Germany) for confocal imaging studies. Fluorescence images were acquired using Zeiss LSM-510 Meta confocal microscope (Zeiss, Germany) with argon/2 excitation laser at 488 nm. Image processing and analysis were conducted with ImageJ software. For intensity analysis equal amount of background subtraction was applied to all images (rolling ball radius: 1000 pixels).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00491. Figure S1 showing confocal microscope images of A549 tumor-like spheroids treated with COOH− and mPEG− Qdots for 24 h (PDF) J


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative (Lower Saxony Centre for Biomedical Engineering, Implant Research and Development in Hannover), which is financially supported by the Lower Saxony ministry of Science and Culture and the Volkswagen Foundation. Part of this work was also funded by the German Research Foundation (DFG) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). R.J. acknowledges the Niedersächsische Krebsgesellschaft e.V. for financial support. We also thank Prof. Dr.-Ing. Birgit Glasmacher and Daniel Mueller (Institute of Multiphase Processes) for providing us access to the confocal microscope, Hamza Belhadj for his assistance in ATR-FTIR analysis, Paul Maschhoff (Department of Chemical Engineering, Northeastern University) for his support in editing the manuscript, and Marc Krey for his assistance in TG analysis.

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